Figure 1.
Experimental design of hepatocyte cultures.
Four different conditions were utilized for the metabolite measurements. Collagen single gel [CSG], collagen double gel [CDG] collagen-soluble Adipogel sandwich [CSG+solASG] and collagen-Adipogel sandwich cultures [CSGASG]. Secreted products were measured at the recovery stage, pre-stable stage and stable stage of culture. Urea and albumin synthesis was determined from day 3 to day 10 of culture.
Table 1.
List of intracellular metabolites for mass balance.
Table 2.
Effect of Adipogel substrate on amino acid and glucose metabolism of recovery stage hepatocyte cultures.
Table 3.
Effect of Adipogel substrate on amino acid and glucose metabolism of pre-stable stage hepatocyte cultures.
Table 4.
Effect of Adipogel substrate on amino acid and glucose metabolism of stable stage hepatocyte cultures.
Figure 2.
Functional Analysis of Rat Hepatocytes In vitro Using Adipogel.
[A] Urea and [B] Albumin Secretion rate of Hepatocytes cultured in five different configurations at a density of 500,000 cells/well in a 12 well plate; CSG corresponds to culture on single collagen gel; CDG corresponds to culture in collagen double gel sandwich configuration; CSG+solASG corresponds to hepatocytes cultured on collagen single gel with soluble Adipogel in the media; CSG+ASG corresponds to culture on collagen single gel with Adipogel overlaid on top. Adipogel was utilized at a 1∶5 ratio with culture media and media was changed on days 0, 1,2,5,7 and 9. While the urea secretion rates are similar for the CDG [positive control] and the CSG+solASG conditions, the albumin secretion rate is significantly higher for the CSG+solASG condition as compared to CDG cultures. * indicates p<0.05 vs. CDG condition.
Table 5.
Summary of results for the CSG+solASG condition on different experimental days.
Figure 3.
Metabolic Network Model for Hepatocyte Cultures.
Arrows indicate direction of reaction assumed in the model. Numbers refer to reaction numbers listed in Table S1 and Table S2. Albumin reaction is not shown for purposes of clarity.
Figure 4.
Metabolic Flux Analysis of Rat Hepatocytes In vitro Using Adipogel.
[A] Recovery stage [B] Pre-stable stage and [C] Stable stage of culture. Hepatocytes cultured in five different configurations at a density of 500,000 cells/well in a 12 well plate; CSG corresponds to culture on single collagen gel; CDG corresponds to culture in collagen double gel sandwich configuration; CSG+solASG corresponds to hepatocytes cultured on collagen single gel with soluble Adipogel in the media; CSG+ASG corresponds to culture on collagen single gel with Adipogel overlaid on top;. Adipogel was utilized at a 1∶5 ratio with culture media and media was changed on days 0, 1,2,5,7 and 9. Metabolic Flux Analysis was performed on [A] recovery stage [B] pre-stable stage and [C] stable stage of culture. MFA results for CDG vs. CSG+solASG conditions are represented in the figures. Note that the fluxes in red correspond to significantly upregulated fluxes [with statistical significance of p<0.05] for the CSG+solASG condition as compared to the CDG condition. Fluxes in blue correspond to significantly downregulated metabolite fluxes.